The overall goal of this assay is to observe individual membrane fusion events driven by neuronal and exocytotic SNARE proteins to understand fundamental mechanisms of neurotransmitter and hormone release. This method can help answer key questions in the membrane fusion field, such as how cargo release occurs through a dynamic fusion pore, and the fate of the pore after fusion. The main advantage of this technique is that distinct stages of vesicle docking, fusion for opening, and cargo release kinetics can be observed for individual events within biochemically defined environments.
Though this method can provide insight into exocytosis, it can also be applied to other membrane-fusion reactions, such as the fusion of envelope viruses with target cell membranes during infection. Generally, individuals new to this method will struggle because many different steps need to be mastered from microfabrication to protein purification and imaging. To begin, prepare a silicone master mold using standard photolithography techniques and the template shown here.
Next, add 100 milliliters of PDMS base and 10 milliliters of the curing agent into a disposable plastic cup, and stir the mixture well. Then, place the cup into a vacuum desiccator. Apply a vacuum, but watch it closely to prevent the mixture from overflowing.
Once degassed, pour a large drop of the degassed PDMS mixture into a glass petri dish, and press the mold onto the PDMS with the template side facing up to avoid trapping bubbles beneath the wafer. Now, pour degassed PDMS onto the template until it is covered by five to eight millimeters of the polymer. If an air bubble occurs, gently remove it with a pipette tip.
Place the PDMS-covered template on the level surface of an oven, and bake it for three hours at 60 degrees Celsius. Then, use a new scalpel blade to cut out a PDMS block containing the molded channel structures. Note that the cut out block needs to fit onto a cover slip.
Next, place the side of the channel grooves up, and use a hole-punch to drill through the block in one continuous motion. Be sure to remove the punched-out piece from each of the eight hole locations. When finished, stick the block grooves down onto a clean piece of aluminum foil.
Now, cut tubing so that it has a slight slant at one end, and use a pair of tweezers to push the tubing about one-third of the way into the punched holes. Cut the tubes so that they are able to reach the buffer reservoir and the syringe pump. To connect the tubing to the syringes on the syringe pump, cut a short piece from larger silicone tubing, and into one side, insert the thinner tube.
This ready-to-use block of PDMS can be kept for a few months. First, place the PDMS block under high vacuum for at least 20 minutes to remove dissolved gases and reduce the risk of air bubbles in the microfluidic channels during the fusion experiment. Next, make sure that the microscope stage area is set to the desired temperature.
While the temperature equilibrates, prepare the NBD-PE fluorescently labeled small unilamellar vesicles containing t-SNARE proteins, or protein-free controls, by diluting 30 microliters of the vesicles with 60 microliters of buffer. Then, draw up the solution into a three-milliliter syringe. Press out most of the air above the sample while holding the syringe vertically.
Then, seal the tip using paraffin film, and create a vacuum by pulling down the plunger. While pulling a vacuum, tap the syringe barrel to accelerate degassing of the solution. Repeat this process a few times until no more air bubbles occur when the vacuum is applied.
Next, use a hypodermic needle with a diameter slightly larger than the microfluidic tubing to punch a hole in the cap of a microcentrifuge tube. Fill the degassed small unilamellar vesicles into the microcentrifuge tube, place it into the holder on the microscope stage, and allow the solution to equilibrate to the set temperature. Place a clean cover slip into a plasma cleaner, and run air plasma over the cover slip for about five minutes.
Then, remove the cover slip and place it, treated side up, on top of a few lint-free tissues serving as a cushion. Next, place the PDMS block, patterned side facing down, on top of the cover slip. Use a pair of tweezers to gently press down on the block, but do not press so hard as to break the glass.
Place the assembled flow cell onto the microscope stage. Then, connect the tubes to the small unilamellar vesicle reservoirs and to the syringe pump. Start aspirating the small unilamellar vesicles at three microliters per minute until the solution fills the channels completely.
When the solutions in all of the channels start moving up the tubing on the exit side, reduce the flow to 0.5 microliters per minute, and incubate for 30 to 45 minutes. The time between plasma-treating the glass cover slip and flowing the SUVs into the channel should not exceed 10 minutes, as the effect of the plasma treatment is transient. Check the channels for any leaks using fluorescence imaging with a low magnification objective, and then, rinse all channels with degassed buffer to wash away unbound vesicles.
Then, switch to the high-magnification oil-immersion objective for TIRF microscopy, and secure the flow cell by taping it onto the microscope stage. Close the field's diaphragm to about 40 micrometers in diameter, and adjust the 488-nanometer excitation light intensity using the software in order to bleach the fluorescence in the exposed area significantly, but not completely. The fluorescence intensity in the middle of the exposed area should be lower than at the edges, as the intact NBD-PE molecules enter and diffuse a certain distance before bleaching.
However, if the surface-adherent SUVs fail to burst, all the fluorophores in the exposed area should bleach. To verify the results of the study state measurements, stop the illumination and restart it a few minutes later to see if the fluorescence has recovered. Degas the reconstitution buffer, and use it to dilute the v-SNARE reconstituted small unilamellar vesicle stock solution, so that the resultant solution produces 10 to 100 fusion events in the field of view over the course of 60 seconds.
Too many fusion events increase the background fluorescence and make detection and analysis of the events difficult. In contrast, a fusion rate that is too low can result in poor statistics. Flow the v-SNARE reconstituted small unilamellar vesicles into the channel at a flow rate of two microliters per minute, and switch to the appropriate excitation emission settings to monitor lipid mixing.
Then, adjust the TIRF angle and polarization as described in the accompanying text protocol. Continuously excite and monitor the vesicles'fluorescence using a 561-nanometer laser. As the vesicles reach the flow channel and dock onto and fuse with the supported bilayer, the background fluorescence signal starts to accumulate.
Adjust the excitation laser intensity through the software to continuously bleach the background fluorescence, so that new docking and fusion events can readily be observed. Acquire several movies at different positions in a given channel. Check NBD-PE fluorescence to verify that the supported bilayer does not have defects at these positions.
The fluorescence recovery after photo bleaching is shown here for a t-SNARE reconstituted supported bilayer that was relatively defect-free and fluid. The area is imaged at low light exposure to follow the fluorescence recovery by diffusion of NBD-labeled lipids. It is easier to observe docking and fusion events while monitoring fluorescence from lipid labels alone.
In the sequence of images shown here, a frame was recorded every 18 milliseconds. Upon fusion, the lipid labels transfer into the supported bilayer and diffuse away from the site of fusion. In some cases, it is desirable to monitor lipid mixing and soluble cargo release simultaneously.
A representative event describing simultaneous lipid mixing and soluble cargo release is shown here. At the high concentrations used for encapsulation, the soluble content fluorescence depicted in red is initially self-quenched. A sharp increase in lipid fluorescence, depicted in blue, is indicative of lipid mixing due to a fusion event as in the single-color monitoring of lipid labels shown earlier.
Pore opening releases soluble content dye and relieves the dye's initial self-quenching in the vesicle lumen. This leads to an increase of the soluble cargo signal, plotted in red. Interestingly, the cargo's fluorescence reached a stable plateau.
This indicates that the pore resealed after releasing all lipid labels and a fraction of the soluble cargo. At the end, the soluble content signal, depicted in red, decreases very rapidly. This is likely due to either rapid full fusion or liposome bursting.
After watching this video, you should have a good understanding of how to prepare a microfluidic flow cell form a supported bilayer reconstituted with t-SNARE proteins, and monitor and analyze single vesicle fusion events using TIRF microscopy. Thanks for watching, and good luck with your experiments.
